Amitriptyline is a potent antagonist of histamine H1 and H2 receptors. High affinity binding of 3H-amitriptyline can be used to specifically label the H1 receptor as shown by KD values of H1 antagonists that agree with those of known H1 systems. These methods are also being adapted to study amitriptyline binding to H2 receptors. The H2 receptor density (Bmax) will be measured in subcellular and regional fractions of guinea pig, rat and mouse brain. In homogenates the H2 but not the H1 receptor is linked to adenylate cyclase; in brain slices both receptors have been implicated. To test the latter, we will study the effect of selective H1 and H2 agonists and antagonists on the histamine activation of cyclic AMP synthesis in brain slices and measure the KD values for the antagonists, the definitive way of identifying the receptors. The same approach will be used to study the receptors that mediate the histamine-activated cyclic GMP synthesis in brain slices. Tricyclic antidepressants are H1 and H2 antagonists but their mode of receptor antagonism may be different from those of the conventional one, e.g., mepyramine and cimetidine. KD values for some of these drugs will be obtained by studying their competition with both 3H-mepyramine and 3H-amitriptyline to the H1 receptor. Since the KD of amitriptyline, but not cimetidine, was increased for the H2-linked adenylate cyclase when guanylylimidophosphate (GppNHp) was substituted for GTP, we will compare the KD values of tricyclic and non-tricyclic H2 blockers obtained in the presence of GTP and GppNHp. Since there is no re-uptake of histamine, its enzymatic methylation may serve as the inactivation mechanism. This will be tested with inhibitors of his-histamine methyltransferase (HMT) which should potentiate the activation of adenylate cyclase by histamine. Results could be complicated by direct actions of these drugs on the receptor, which will be determined by measuring their effects on the activation by dimaprit, an H2 agonist that is not a substrate for HMT. Since all evidence indicates that formation of methylhistamine in brain depends on histamine release, methylhistamine content may reflect the functioning of histaminergic nerves. The action of some drugs known to affect this system will be studied on the reglonal content of histamine and methylhistamine, the latter measured by a method recently developed by us.